EP1264176A1 - Mesure de changements d'activite metabolique - Google Patents

Mesure de changements d'activite metabolique

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Publication number
EP1264176A1
EP1264176A1 EP01917974A EP01917974A EP1264176A1 EP 1264176 A1 EP1264176 A1 EP 1264176A1 EP 01917974 A EP01917974 A EP 01917974A EP 01917974 A EP01917974 A EP 01917974A EP 1264176 A1 EP1264176 A1 EP 1264176A1
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Prior art keywords
gas
organism
germination
metabolic
oxygen
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EP01917974A
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German (de)
English (en)
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EP1264176B1 (fr
Inventor
Albert Van Duijn
Johan Willem KÖNIG
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Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
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Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
    • G01N33/4977—Metabolic gas from microbes, cell cultures or plant tissues

Definitions

  • the invention relates to methods for measuring metabolic states, metabolic rates and changes therein, such as growth rates, dying rates, cell division, metabolite production, and other biological activities of organisms, in particular of small multi-cellular organisms, of seeds and seedlings and of micro-organisms, such as fungi, yeast, bacteria, plant or animal cells and cultures thereof.
  • Biological activities of organisms are manifold and are often studied by the chemical, physical, physiological or morphological ways they manifest themselves. Culturing organisms, be it micro-organisms in cell culture, plants, or others, requires managing these biological activities, and managing these activities often requires measuring the underlying metabolic activities. As an example herein germination of seeds is discussed, however, the invention extends to culturing other organisms where similar approaches apply.
  • a new plant formed by sexual reproduction starts as an embryo within the developing seed, which arises from the ovule.
  • the seed When mature, the seed is the means by which the new individual is dispersed, though frequently the ovary wall or even extrafloral organs remain in close association to form a more complex dispersal unit as in grasses and cereals.
  • the seed therefore, occupies a critical position in the life history of the higher plant.
  • the success with which the new individual is established -the time, the place, and the vigour of the young seedling- is largely determined by the physiological and biochemical features of the seed.
  • Germination begins with water uptake by the seed (imbibition) and ends with the start of elongation by the embryonic axis, usually the radicle. It includes numerous events, e.g., protein hydration, subcellular structural changes, respiration, macromolecular syntheses, and cell elongation, none of which is itself unique to germination. But their combined effect is to transform an organism having a dehydrated, resting metabolism into an organism having an active metabolism, culminating in growth.
  • Germination sensu stricto therefore does not include seedling growth, which commences when germination finishes. Hence, it is incorrect, for example, to equate germination with seedling emergence from soil since germination will have ended sometime before the seedling is visible. Seed testers often refer to germination in this sense because their interests lie in monitoring the establishment of a vigorous plant of agronomic value. However, physiologists do not encourage such a definition of the term germination but in general acknowledge its widespread use by seed technologists. It would however be preferable to find a more defined definition. Processes occurring in the nascent seedling, such as mobilisation of the major storage reserves, are also not part of germination: they are postgermination events.
  • a seed in which none of the germination processes is taking place is said to be quiescent.
  • Quiescent seeds are resting organs, generally having a low moisture content (5-15%) with metabolic activity almost at a standstill. A remarkable property of seeds is that they are able to survive in this state, often for many years, and subsequently resume a normal, high level of metabolism.
  • quiescent seeds generally need only to be hydrated under conditions that encourage metabolism, e.g., a suitable temperature and presence of oxygen. Components of the germination process, however, may occur in a seed that does not achieve radicle emergence.
  • Dormant seeds are converted into germinable seeds (i.e., dormancy is broken) by certain "priming" treatments such as a light stimulus or a period at low or alternating temperature which nullify the block to germination but which themselves are not needed for the duration of germination process.
  • the extent to which germination has progressed can be determined roughly, say by measuring water uptake or respiration, but these measurements give us only a very broad indication of what stage of the germination process has been reached. No universally useful biochemical marker of the progress of germination has been found. The only stage of germination that we can time fairly precisely is its termination.
  • Emergence of the axis (usually the radicle) from the seed normally enables us to recognise when germination has gone to completion, though in those cases where the axis may grow before it penetrates through the surrounding tissues, the completion of germination can be determined as the time when a sustained rise in fresh weight begins.
  • Germination behaviour of large numbers of seeds, e.g., all the seeds produced by one plant or inflorescence, or all those collected in a soil sample, or all those subjected to certain experimental treatment.
  • the degree to which germination has been completed in a population is usually expressed as a percentage, normally determined at time intervals over the course of the germination period which can be expressed in so-called germination curves, about which some general points should be made.
  • Germination curves are usually sigmoidal, a minority of the seeds in the population germinates early, then the germination percentage increases more or less rapidly, and finally few late germinatores emerge.
  • curves are often positively skewed because a greater percentage germinates in the first half of the germination period than in the second. But although the curves have the same general shape, important differences in behaviour between populations are evident. For example, curves often flatten off when only a low percentage of the seeds has germinated, showing that this population has low germination capacity, i.e., the proportion of seeds capable of completing germination is low. Assuming that these seeds are viable, the behaviour of the population could be related to dormancy or to environmental conditions, such as temperature or light, which do not favour germination of most of the seeds.
  • the shape of the curves also depends on the uniformity of the population, i.e., the degree of simultaneity or synchrony of germination. When a limited percentage of seeds succeeds in germinating fairly early, but the remainder begin to do so only after a delay the population seems to consists of two discrete groups: the quick and the slow germinators. This example also illustrates the point that populations with the same germination capacity can differ in other respects.
  • glycolysis catalysed by cytoplasmic enzymes, operates under aerobic and anaerobic condition to produce pyruvate, but in the absence of O 2 this is reduced further to ethanol, plus CO2, or to lactic acid if no decarboxylation occurs.
  • Anaerobic respiration also called fermentation, produces only two ATP molecules per molecule of glucose respired, in contrast to six ATPs produced during pyruvate formation under aerobic conditions.
  • oxidative decarboxylation of pyruvate produces acetyl-CoA, which is completely oxidised to CO2 and water via the citric acid cycle to yield up to a further 30 ATP molecules per glucose molecule respired.
  • the generation of ATP molecules occurs during oxidative phosphorylation when electrons are transferred to molecular O2 along an electron transport (redox) chain via a series of electron carriers (cytochromes) located on the inner membrane of the mitochondrion.
  • redox electron transport
  • cytochromes electron carriers located on the inner membrane of the mitochondrion.
  • An alternative pathway for electron transport which does not involve cytochromes, may also operate in mitochondria.
  • the pentose phosphate pathway is an important source of NADPH, which serves as a hydrogen and electron donor in reductive biosynthesis, especially of fatty acid.
  • Intermediates in this pathway are starting compounds for various biosynthetic processes, e.g., synthesis of various aromatics and perhaps nucleotides and nucleic acid.
  • complete oxidation of hexose via the pentose phosphate pathway and the citric acid cycle can yield up to 29 ATPs.
  • Keto acids e.g.; ⁇ -ketoglutarate, pyruvate
  • Phase 1 Initially there is a sharp increase in O2 consumption, which can be attributed in part to the activation and hydration of mitochondrial enzymes involved in the citric acid cycle and electron transport chain. Respiration during this phase increases linearly with the extent of hydration of the tissue.
  • Phase 2. This is characterised by a lag in respiration as O2 uptake is stabilised or increases only slowly. Hydration of the seed parts is now completed and all pre-existing enzymes are activated. Presumably there is little further increase in respiratory enzymes or in the number of mitochondria during this phase. The lag phase in some seeds may occur in part because the coats or other surrounding structures limit O2 uptake to the imbibed embryo or storage tissues, leading temporarily to partially anaerobic conditions.
  • Phase 4 This occurs only in storage tissues and coincides with their senescence following depletion of the stored reserves.
  • the lengths of phases 1-4 vary from species to species owing to such factors as differences in rates of imbibition, seed-coat permeability to oxygen, and metabolic rates. Moreover, the lengths of the phases will vary considerably with the ambient conditions, especially the temperature. In a few seeds, e.g., Avena fatua, there is no obvious lag phase in oxygen uptake. The reasons for its absence are not known, but it could be because efficient respiratory systems become established early following imbibition, including the development of newly active mitochondria, thus ensuring a continued increase in O2 utilisation. Also, coat impermeability might not restrict O2 uptake prior to the completion of germination.
  • raffinose- series oligosaccharides raffinose (galactosyl sucrose), stachyose (digalactosyl sucrose), and verbascose (trigalactosyl sucrose), although the latter is usually present only as a minor component.
  • raffinose galactosyl sucrose
  • stachyose digalactosyl sucrose
  • verbascose trigalactosyl sucrose
  • sucrose and the raffinose-series oligosaccharides are hydrolysed, and in several species the activity of ⁇ -galactosidase, which cleaves the galactose units from the sucrose, increases as raffinose and stachyose decline.
  • ⁇ -galactosidase which cleaves the galactose units from the sucrose
  • Free fructose and glucose may accumulate in seeds during the hydrolysis of sucrose and the oliosaccharides, but there is no buildup of galactose (e.g., in mustard, Sinapis alba). Hence, it is probably rapidly utilised, perhaps through incorporation into cell walls or into galactolipids of the newly forming membranes in the cells of developing seedling.
  • the above example illustrates an archaic way of measuring the underlying metabolic activity of an organism. More modern methods have been developed which comprise measuring oxygen or other metabolic gasses in gas or liquid media. Oxygen, or other gasses, in gas are often measured by analysis with gas-chromatography. In liquid gas contents are often measured by flushing some liquid through an electro-chemical measurement device.
  • the present invention recognizes this problem and provides a method for determining metabolic state or rate or a change therein of at least one organism or part thereof comprising placing said organism or part thereof in a confined container and measuring the concentration of a metabolic gas in said confined container to determine consumption or production of said gas by said organism or part thereof wherein said gas concentration is determined without essentially affecting the concentration of said gas in said confined container.
  • Such a method according to the invention has multiple advantages, for example that the equilibrium of the gases within the confined container is not disturbed or influenced because the said container does not have to be opened to take a sample, thereby providing a very accurate and reliable method to determine the concentration of a metabolic gas in said confined container and as a consequence the metabolic state or rate or a change therein caused by at least one organism or part thereof is accurate and reliably determined.
  • the invention provides a method for determining the metabolic state of at least one organism or part thereof comprising placing said organism or part thereof in a confined container and measuring the concentration of a metabolic gas in said confined container to determine consumption or production of said gas by said organism or part thereof wherein said gas concentration is determined without essentially affecting the concentration of said gas in said confined container. If no change in metabolic gasses are detected (in practice for a sufficiently long period), it may for example be assumed that the organism is dead or in a hibernating state, in particular now where the invention provides that no gas is consumed by measuring, all changes in gas concentration must thus be attributed to the production and/or consumption of a metabolic gas, thus of life, or at least in a state of life-like activity.
  • An example of an organism as disclosed herein within the experimental part is a seed or a worm. It is clear to a person skilled in the art that different organisms or parts thereof are tested by a method according to the invention as long as the organism or part thereof fits within a confined container. Therefor a method according to the invention is performed in a confined container which may have different sizes and/or shapes depending on the organism or part thereof which need to be studied.
  • An example of a part of an organism are the roots of a plant. The experimental part describes a rose from which the roots were put in a confined container.
  • Another example of a part of an organism is a cell or a cell culture. Methods to arrive at a proper cell or cell culture are well known by the person skilled in the art.
  • a method according to the invention is used to determine changes in gas concentration of a predetermined organism or part thereof. Changes can therefor be attributed to a known, predetermined organism or part thereof.
  • metabolic gases from which the changes in concentration can be determined are oxygen, carbon dioxide, carbon mono-oxide, nitric oxide, nitric dioxide, dinitric oxide, ethylene and ethanol. All these gases can be measured with different organo-metal complexes.
  • a confined container (also called confined space; the terms may be used interchangeably herein) is herein defined as a container that is properly shut to (essentially) avoid gas exchange between the confined container and the surrounding and furthermore a confined container is defined as a container that is essentially not opened during measurements but to which additional substances (oxygen, nutrients, growth hormones, etc.) can be added with for example a valve or injection system. Because the container is essentially not opened all changes in a metabolic gas concentration are attributed to the metabolic state or rate or a change therein of the organism or part thereof which is located in the container.
  • a confined container has different shapes and/or sizes dependent on the organism or part thereof studied.
  • the invention provides a method for determining a change in metabolic state or rate of at least one organism or part thereof comprising placing said organism or part thereof in a confined container and repeatedly or continually measuring the concentration of a metabolic gas in said container to determine changes in consumption or production of said gas by said organism or part thereof wherein said gas concentration is determined without essentially affecting the concentration of said gas in said confined container.
  • one or more seeds are brought in a small confined container, along with some water to induce the germination process. Seeds can of course be totally immersed in water, which typically allows for measurements to be made in the liquid but usually measurement of the air or gas above the seeds will be sufficient.
  • the seed(s) Due to the germination at a certain point in time the seed(s) will start to consume oxygen and produce carbon dioxide.
  • the oxygen concentration will drop from the moment the germination starts and the carbon dioxide concentration will rise.
  • the gas concentration is preferably measured optically. This can for example be achieved by a measuring device which is at least partly set up within the confined container, but measurements can also be made through a clear portion of the wall of the confined container, which for example could be made of glass.
  • the invention provides an optical method based on fluorescence quenching of fluorescent compounds by oxygen (1,2,3,4), to determine the oxygen levels inside a container, preferably without opening it.
  • a sample can be measured over and over again in the time, and is not destroyed. Moreover, because the sample is not destroyed the number of samples necessary to do a time study is considerably lower compared to conventional methods.
  • the invention provides a method wherein said gas concentration is determined by determining the fluorescence quenching of a fluorescent dye, preferably a suitable organo- metal, present in said confined container.
  • an oxygen sensitive dye such as a ruthenium bipyridyl complex, or Tris-Ru 2+ 4,7 biphenyl 1,10 phenantrolin; or another Ru(ruthenium)-complex, or another organo- metal complex, such as an Os-complex or a Pt-complex, is suitable, for measuring carbon dioxide, or other gasses such as CO, NO, NO2, N2O, ethylene or ethanol, suitable sensitive organo-metal dyes, such as tris[2-(2- pyrazinyl)thiazole] ruthenium II (5) are used.
  • the optical oxygen sensing measurement technique used herein is based on the fluorescence quenching of a metal organic fluorescent dye.
  • the dye which is very sensitive to oxygen is for example excited by a short laser light-pulse of for example 1 microsecond. After the excitation has stopped the oxygen sensitive dye emits fluorescent light with a decay curve which depends on the oxygen concentration. The process behind this phenomenon is called dynamic quenching.
  • said dye is present in a gas permeable compound such as silica or a hydrophobic polymer such as a (optionally fluoridated) silicone polymer, in PDMS (polydimethylsiloxane), in PTMSP (polytrimethylsilylpropyl), or in a mixture thereof but of course it can be contained in other suitable compounds as well.
  • a gas permeable compound such as silica or a hydrophobic polymer
  • PDMS polydimethylsiloxane
  • PTMSP polytrimethylsilylpropyl
  • the invention provides a method wherein said dye is present in at least a part of an inner coating of said confined container, for example situated on the inside of an optically transparent part of the confined container when measurement is from the outside.
  • Measuring can for example be achieved by measuring the fluorescence lifetime.
  • the excited molecules are deactivated by oxygen in a collision process.
  • the quenching process does not consume the gas (here the oxygen) so liquid medium does not necessarily have to be stirred to obtain the measurements.
  • the fluorescence lifetime gets shorter because the probability of the molecules to be deactivated gets higher for molecules which stay longer in the excited state. The effect is proportional with the quencher concentration.
  • the relation between fluorescence lifetime and gas (here oxygen) concentration is given by the Stern Volmer equation (1)
  • ⁇ 0 is the fluorescence lifetime at quencher (O2) concentration zero
  • ⁇ is the fluorescence lifetime at a specific quencher (O2) concentration
  • Csv is the Stern- Volmer constant
  • [O2] is the gas concentration.
  • Measuring can also be achieved by measuring the fluorescence intensity.
  • the fluorescent compound is excited by a continuously radiating light source such as a LED and the fluorescence intensity is measured. More gas (here oxygen) caused less fluorescence.
  • More gas here oxygen caused less fluorescence.
  • the relation between the oxygen concentration and the intensity is given by the Stern Volmer equation (2)
  • I 0 is the fluorescence intensity at quencher (O2) concentration zero
  • I is the fluorescence intensity at a specific quencher (O 2 ) concentration
  • Csv is the Stern-Volmer constant and [O2] is the gas concentration.
  • the fluorescence lifetime method has the advantage that the measurement is independent of the source intensity, detector efficiency, fluorescent probe concentration etc.
  • a method based on this principle is robust and less prone to drift.
  • the method as provided by the invention is very useful to measure metabolic rate changes of organisms by measuring an increase or decrease in metabolic gas production or consumption by said organism or organisms.
  • a method as provided by the invention is based on a time gated measurement (Fig.l).
  • the fluorescence is determined in two time windows (A and B) after a light pulse. Fluorescence lifetime is a function of the ratio between A and B and is proportional to the oxygen concentration.
  • Figure 2 shows an example of a simplified experimental set-up.
  • the confined container (having possibly different sizes and/or shapes) contains an oxygen sensitive coating situated on the inside of an optically transparent part of the confined container. Another possibility is to provide the oxygen sensitive substance to the material from which the confined contain is made.
  • oxygen sensitive substance via a holder at any desired position within the confined container.
  • the oxygen sensitive substance can be placed at every desired position as long as it is possible to reach the position with for example a laser to provoke excitation and to determine the fluorescence signal with a detector. Detection of the fluorescence is made visible by for example a measuring device or with help of a computer and suitable computer programs.
  • the confined container is not physically part of the measuring device, but is clear that it possible to set-up a measuring device which is partly set-up in a confined container.
  • a confined container can have different shapes and/or sizes and can be made of different materials as long as it is possible to perform measurements through a clear portion of the wall of the confined container, which for example could be made of glass. As described it is also possible that part of measuring device is part of the confined container in which case it is not necessary for the wall of the container to be clear.
  • Figure 3 shows a more detailed instrumental set up.
  • a light source e.g. LED or laser
  • the light pulses are filtered and excite the fluorescent dye located in the environment where the metabolic gas has to be determined.
  • the resulting fluorescence response is detected in a detector, the information is digitised, if needed the measurement is corrected (for temperature for example) and the gas concentration is calculated and displayed.
  • Oxygen consumption measurements on seeds during germination and priming are important for the following reasons.
  • quality of seed batches both for use as plant propagation method in e.g. horticulture and in industrial applications in e.g. barley malting
  • the following aspects that can be achieved by measuring oxygen consumption during germination are important: (i) speed of germination, (ii) homogeneity of germination of a seed batch, (iii) monitoring system that is automated and (iv) possibility to measure large numbers of individual seeds.
  • a method according to the invention is as well applicable to register a second respiratory burst as is often identified in phase 3 of germination.
  • the invention is furthermore used for quality assurance. For example seed batches primed or germinated by different methods or under different circumstances are tested, varieties are tested on for example their germination.
  • a method according to the invention is preferably miniaturised and/or automated. An example of such a automated/miniaturised device is depicted in Figure 7.
  • a preferably automated, quality assurance and/or high throughput screening is also used on another organism or part thereof.
  • said organism or part thereof comprises one or more micro-organisms or part thereof such as a protoplast, plastid (e.g.
  • chloroplast or mitochondrium
  • the invention provides a method wherein said change in metabolic rate denotes cell activity of said organism or part thereof or micro-organism or cultures thereof, or, alternatively, wherein said change in metabolic rate denotes cell death, and to detect circumstances wherein such cell-activities thrive, or not.
  • a method according to the invention is for example useful to detect ( the onset of) sporulation of bacterial cultures, or microbial fermentation.
  • the invention provides a method to determine or monitor a rate of seed germination or development, to for example determine proper priming of seed batches. Therewith, the invention also provides a seed batch monitored with a method according to the invention. Said seed batches have accurately been primed.
  • the invention provides a method to determine or monitor a rate of culture development of a cell- or tissue-culture comprising use of a method according to the invention and a cell- or tissue culture monitored with a method according to the invention.
  • Other methods provides for example entail a method to determine or monitor processing of waste water comprising use of a method according to the invention, or other processes where micro-biological fermentation plays a role.
  • the invention is also used to determine the oxygen consumption of other organisms such as micro organisms, animals, such as e.g. an insect or a worm.
  • This part of the invention is e.g. useful to determine the presence of wood worms in a piece of (antique) wooden furniture or to determine the presence of wood worms in for example the wooden foundation or wooden floors in a house.
  • a method according to the invention is also used to test the effect of for example an insecticide on its target by placing one or more targets in a confined space and determining the metabolic state or rate or a change therein and compare with one or more target(s) not treated with the insecticide.
  • the tested and control targets have been selected on for example their oxygen consumption, thereby providing good control experiments.
  • Different analogues or derivatives of an insecticide are for example tested for their effectiveness.
  • One or more seeds are brought in a confined container, along with some water to induce the germination process.
  • the container is closed. Due to the germination at a certain point in time the seed(s) will start to consume oxygen. Because the container is closed, the oxygen concentration will drop from the moment the germination starts. This can be monitored with a special oxygen sensitive coating on the inside of an optically transparent part of the container.
  • An advantage of optical oxygen determination is the fact that the coating itself does not consume any oxygen. In this way the start of the germination can be monitored accurately. Up to now only the appearance of a root was an indication of the germination. In the experiment the first root showed after 10 to 14 hours. From the oxygen measurements we see that the germination activity showed after 3.5 hours. The oxygen consumption is an early indicator of seed germination.
  • the point where the germination starts can be calculated from the measured oxygen levels, as for example given in table 1.
  • the linear extrapolation of the oxygen levels measured after 4 hours in the containers with 1, 2 and 3 seeds show an intersection with the oxygen level of an empty container at 3.5 hours after the addition of the water. This is the point in time where the metabolism of the germination starts. This is shown in figure 4.
  • Figure 5 shows a general course of oxygen levels with seeds in a container and figure 6 an example of a calculation.
  • Table 1 Oxygen content in ⁇ g of a sample container (approx. 1 ml) with a different number of seeds.
  • the oxygen consumption of plant roots is measured by placing the roots of a plant in a confined container with a known volume sealed hermetically around the stem in order to avoid gas exchange of the confined container and surrounding.
  • the oxygen consumption profile of the plants under different growth conditions can be easily determined.
  • Figure 8 shows a schematic representation of the experimental set-up of a plant (for example a rose) in a confined container.
  • FIG. 9 shows the result. In this example two different kinds of metabolisms were found, depending on the oxygen concentration.
  • Figure 7 shows a schematic set-up of the confined containers for this experiment.
  • the use of small confined containers as in this example shows that a method according to the invention is easily miniaturised.
  • Figure 10 shows the result of the above described experiment. This result is in accordance with the visual determination of germination of the 2 tested seeds.
  • the oxygen consumption of different worms was also determined with the Non Invasive Oxygen Detection (NIOD) Method.
  • NIOD Non Invasive Oxygen Detection
  • Figure 11 shows a confined space comprising a worm and Figure 12 shows the results of the oxygen consumption of 2 different kinds of worms. It was calculated that the wood worm of 93 mg consumed about 0.5 microgram oxygen per minute. This information was used to optimise the non-toxic killing method of wood worms in houses.
  • Figure 2 A rough schematic representation of a set-up for measuring metabolic gas changes and/or rates.
  • FIG. 3 A detailed schematic representation of oxygen sensor.
  • Figure 5 Oxygen consumption during the germination of seeds.
  • Figure 7 Example of an automated version of a method according to the invention.
  • Figure 10 Determination of the start of germination by oxygen consumption in a confined space.
  • Figure 11 Picture showing a confined space comprising a worm

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Abstract

L'invention concerne des procédés destinés à mesurer des états, ou des activités ou des changements métaboliques, tels que vitesse de croissance, taux de mortalité, division cellulaire, production de métabolite, et d'autres activités biologiques d'organismes, en particulier de petits organismes multicellulaires, tels que des champignons, des levures, des bactéries, des cellules végétales ou animales ainsi que de leurs cultures. L'invention concerne un procédé permettant de déterminer un changement d'activité métabolique d'au moins un organisme qui consiste à enfermer cet organisme ou une partie de cet organisme dans un contenant et à mesurer de manière continue ou répétée la concentration d'un gaz métabolique dans ce contenant afin de déterminer les changements de consommation ou de production du contenant et d'obtenir les changements en consommation ou production de ce gaz par l'organisme, la concentration de ce gaz étant déterminée sans affecter sensiblement sa concentration dans le contenant.
EP01917974A 2000-03-17 2001-03-16 Mesure de changements d'activite metabolique Expired - Lifetime EP1264176B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP01917974A EP1264176B1 (fr) 2000-03-17 2001-03-16 Mesure de changements d'activite metabolique

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP00200990A EP1134583A1 (fr) 2000-03-17 2000-03-17 Mesure de changements du métabolisme
EP00200990 2000-03-17
EP01917974A EP1264176B1 (fr) 2000-03-17 2001-03-16 Mesure de changements d'activite metabolique
PCT/NL2001/000217 WO2001069243A1 (fr) 2000-03-17 2001-03-16 Mesure de changements d'activite metabolique

Publications (2)

Publication Number Publication Date
EP1264176A1 true EP1264176A1 (fr) 2002-12-11
EP1264176B1 EP1264176B1 (fr) 2005-01-19

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EP00200990A Withdrawn EP1134583A1 (fr) 2000-03-17 2000-03-17 Mesure de changements du métabolisme
EP01917974A Expired - Lifetime EP1264176B1 (fr) 2000-03-17 2001-03-16 Mesure de changements d'activite metabolique

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EP00200990A Withdrawn EP1134583A1 (fr) 2000-03-17 2000-03-17 Mesure de changements du métabolisme

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US (1) US20040033575A1 (fr)
EP (2) EP1134583A1 (fr)
AT (1) ATE287537T1 (fr)
AU (2) AU2001244846B2 (fr)
CA (1) CA2403253C (fr)
DE (1) DE60108480T2 (fr)
ES (1) ES2236202T3 (fr)
NZ (1) NZ521622A (fr)
WO (1) WO2001069243A1 (fr)

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Also Published As

Publication number Publication date
WO2001069243A1 (fr) 2001-09-20
DE60108480T2 (de) 2006-03-23
NZ521622A (en) 2004-06-25
CA2403253A1 (fr) 2001-09-20
ES2236202T3 (es) 2005-07-16
DE60108480D1 (de) 2005-02-24
AU2001244846B2 (en) 2005-10-06
AU4484601A (en) 2001-09-24
US20040033575A1 (en) 2004-02-19
EP1264176B1 (fr) 2005-01-19
EP1134583A1 (fr) 2001-09-19
CA2403253C (fr) 2011-02-08
ATE287537T1 (de) 2005-02-15

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